The human immunodeficiency virus type 1 (HIV-1) accessory protein, Vpr, interacts with several host cellular proteins including uracil DNA glycosylase-2 (UNG2) and a cullin-RING E3 ubiquitin ligase assembly (CRL4 DCAF1 ). The ligase is composed of cullin 4A (CUL4A), RING H2 finger protein (RBX1), DNA damage-binding protein 1 (DDB1), and a substrate recognition subunit, DDB1-and CUL4-associated factor 1 (DCAF1 The viral protein R (Vpr) 2 is one of four HIV-1 accessory proteins (Nef, Vif, Vpr, and Vpu), that regulate virus infectivity, primarily through interactions with host proteins (1). Vpr is highly conserved in HIV-1, HIV-2, and the simian immunodeficiency viruses (2-4). Several biological roles for Vpr during viral infection of cells have been described, including facilitation of nuclear translocation of preintegration complexes (5-7), modulation of mutation frequency (8, 9), induction of cell cycle arrest in the G 2 /M phase (10 -15), and stimulation of host cell apoptosis (16 -18). More recently, Vpr has been postulated to enhance infection by mediating the degradation of unknown cellular defense factors (1, 19).To date, three of the four HIV-1 accessory proteins, including Vpr, have been found to interact with cullin-RING finger E3 ubiquitin ligases (CRLs). E3 ligases are multisubunit complexes that include a cullin (CUL), a RING H2 finger protein (RBX1), an adaptor, and a substrate recognition subunit (20). More specifically, Vpr interacts with the CRL4 DCAF1 E3 ubiquitin ligase, assembled with cullin 4A (CUL4A), RBX1, DDB1 (DNA damage-binding protein 1), and DCAF1 (DDB1-and CUL4-associated factor 1) (1, 21). The substrate recognition subunit of this CRL4, DCAF1, previously known as Vpr-binding protein (VprBP), was originally identified via co-precipitation with Vpr (22). At the present time, substantial evidence suggests that Vpr usurps CRL4 DCAF1 E3 ubiquitin ligases to ubiquitinate (ubiquitylate) and degrade unknown cellular proteins required for cell cycle progression (23-31). In fact, Vpr was reported to bind and modulate the activity of cell cycle-related proteins, such as CDC25 (32), WEE1 kinase (33), and SAP145 (34, 35). In addition, Vpr activates ATM and Rad3-related checkpoint kinase (ATR)-dependent DNA damage signaling pathways, including phosphorylation of the histone 2A variant-X and BRCA1 (36, 37). However, whether Vpr interactions with these proposed host cellular factors result in their degradation via CRL4 DCAF1-Vpr E3 ubiquitin ligases had not been established.One potential target of Vpr is uracil-DNA glycosylase-2 (UNG2), which removes uracil lesions from single-stranded and double-stranded DNA in the base excision repair pathway. Initially identified in a yeast two-hybrid screen (38), UNG2 has been implicated as a Vpr-dependent substrate of the CRL4 E3 ubiquitin ligase (39,40).However, a number of studies investigating the roles of UNG2 in HIV replication resulted in opposing views regarding HIV biology. For example, some studies found that virion-associated UNG2 modulates the innate ...

The cullin4A-RING E3 ubiquitin ligase (CRL4) is a multisubunit protein complex, comprising cullin4A (CUL4), RING H2 finger protein (RBX1) and DNA damage-binding protein 1 (DDB1). Proteins that recruit specific targets to CRL4 for ubiquitination (ubiquitylation), bind the DDB1 adaptor protein via WD40 domains. Such CRL4 substrate recognition modules are DDB1- and CUL4-associated factors (DCAFs). Here we show that for DCAF1, oligomerization of the protein and the CRL4 complex occurs via a short helical region (residues 845–873) N-terminal to DACF1’s own WD40 domain. This sequence was previously designated as a LIS1 homology (LisH) motif. The oligomerization helix contains a stretch of four Leu residues, which appear to be essential for alpha-helical structure and oligomerization. In vitro reconstituted CRL4-DCAF1 complexes (CRL4DCAF1) form symmetric dimers as visualized by electron microscopy (EM) and dimeric CRL4DCAF1 is a better E3 ligase for in vitro ubiquitination of the UNG2 substrate compared to a monomeric complex.